Titel deutsch:
Analyse der SDF‑1a Expression in hMSC für die experimentelle Untersuchung der Chemotaxis von hämatopoietischen Stammzellen
Titel englisch:
Analysis of SDF‑1a expression in hMSC for the experimental
investigation of hematopoietic
stem cell chemotaxis
Autoren:
Christina Leinweber
Rainer Saffrich
Anthony D. Ho
Michael Grunze
Axel Rosenhahn
Institutes:
Angewandte Physikalische Chemie, Universität Heidelberg
Innere Medizin V, Hämatologie und Onkologie, Universitätsklinikum Heidelberg
Institut für Funktionelle Grenzflächen (IFG), Karlsruhe Institute of Technology (KIT)
Introduction: Human mesenchymal stromal cells
(hMSC) can be used as a niche model
for the experimental investigation of the chemotactic migration of human hematopoietic
stem and progenitor cells (HSC) towards their stem cell niche, the so called
homing process. These bone marrow derived hMSC express the messenger chemokine stromal cell-derived factor‑1alpha
(SDF‑1a). Because the exact sensing mechanism and
migration behavior of HSC is still not completely unraveled, we studied
migration of human HSC under laboratory conditions using several chemotaxis
experiments with increasing intricacy from single well up to microfluidic devices.
Methods: For the
definition of chemical gradients inside these setups we explored in detail the
expression of the chemokine SDF‑1a in hMSC applying ELISA tests (RayBiotech). The expression
behavior has been studied dependent on
culture time, cell amount and culture media. Additionally we performed intracellular
immunofluorescence staining of SDF‑1a in hMSC to support the ELISA results. Human HSC
(CD34+) for migration experiments were derived from cord blood or mobilized
peripheral blood. Both, HSC and hMSC, as well as recombinant SDF‑1a were used to investigate HSC
chemotaxis in microwell, transwell and microfluidic migration setups. Results:
The media test clearly shows that SDF‑1a expression in hMSC is strongly dependent on
culture conditions. Highest expression rates and values were obtained within a
commercial MSC culture medium (Lonza). Therein SDF‑1a expression increases with increasing
cell amount and longer culture time. About 24 hours after a medium exchange an
equilibrium plateau concentration in the medium supernatant (about 200pg/ml for
50.000 cells) is reached in most cases. The chemotactic response of HSC
correlates with the expected development of chemokine gradients according to
the measured production rates and diffusion profiles inside the applied
microwell and microfluidic setups. Conclusions: With our study we
demonstrate the importance of hMSC culture conditions in planning in-vitro
chemotaxis and homing experiments with hMSC as a niche model. SDF‑1a expression data measured with ELISA
can be used to calculate chemical gradients inside of microfluidic setups. Thus
it is possible to determine sensing thresholds and sensitivity of HSC in
response to SDF‑1a. Furthermore we showed the applicability of a simple microfluidic setup
in experimental medical research.
Disclosure: No
conflict of interest disclosed.